Ash1p is a nuclear protein found in budding yeast that is an intrinsic factor responsible for generating cell diversity. An example of this process is asymmetric cell division, a significant developmental event that occurs in organisms ranging from bacteria, yeast, worms, and mammals. A major function of Ash1p in Saccharomyces cerevisiae is that of a transcriptional repressor. This protein is preferentially localized to the daughter nucleus following nuclear division. Ash1p actively inhibits transcription of the gene encoding for the HO endonuclease that, in turn, is responsible for mating type switching.While many details have been defined for the mechanism leading to the asymmetric localization of Ash1p, lithe has been learned about its function as a transcriptional repressor. Therefore, the overall objective of the proposal is to understand both the regulation and mechanism of transcriptional repression by Ash1p. To address these goals the first aim of this proposal will utilize yeast molecular and classical genetics to search for proteins that may be required for Ash1p function, along with the yeast two-hybrid system to probe for other proteins that may interact with Ash1p. Aim 2 will utilize chromatin immunoprecipitation assays to define the environment and components that exist at the HO gene when it is being repressed. And aim 3 will examine the chromatin structure of the HO locus in the presence of Ash1p.